Quantifying Phase Behavior and Material Properties of Multicomponent Biomolecular Condensates

Ribonucleoprotein granules, such as stress granules, P bodies, and the nucleolus, are fluid-like sub-cellular condensates formed through liquid-liquid phase separation of multivalent proteins in association with cellular nucleic acids. Here we study how variations in RNA-to-protein stoichiometry modulate the phase behavior and condensed phase fluid dynamics. We find that RNA mediates a protein reentrant phase transition that is marked by a condensation and a subsequent de-condensation process in response to the change in RNA-to-protein ratio at a fixed protein concentration. Employing particle tracking using video microscopy and optical-tweezer induced active droplet fusion, we show that the viscosity and surface tension of protein-RNA condensates are not fixed, but vary across the reentrant phase space. In parallel, we use fluorescence correlation spectroscopy (FCS) to quantify protein and nucleic acid diffusion in the condensed phase. Results of our experiments reveal that the mixture composition plays a fundamental role not only in the formation and dissolution of multi-component heterotypic condensates, but also in controlling their material properties.